The mitochondrial membrane potential (ΔΨm) plays a crucial role in the production of ATP as an energy source of the cell. The electron transport chain (complex I, II, III, IV) positioned at the mitochondrial inner membrane generates this electrochemical potential gradient across the inner membrane by pumping protons through the mitochondrial inner membrane while sequentially transporting electrons through the complexes. This proton gradient is utilized by ATP synthase (complex V) to synthesize ATP from ADP and inorganic phosphate. This cycle can remain functional and constantly produces ATP to sustain the cell only when the electrochemical proton gradient is maintained at a constant level with enough available ADP. ΔΨm is the key component of this electrochemical potential gradient.
Mitochondria are known to regulate cell life and death through control of apoptosis, through a critical, irreversible step involving the mitochondrial permeability transition pore (mPTP), a megapore complex triggered to open under certain conditions at both the mitochondrial inner and outer membrane. Once opened, the permeability of the mitochondrial inner membrane increases drastically, causing the release of bioactive proteins including cytochrome C and the inflow of protons, resulting in an irreversible collapse of the mitochondrial membrane potential. This process is known to lead to apoptosis or cell death. In addition, malfunctions and abnormal behaviors of mitochondria are highly associated with the degenerative diseases and the aging process.
To date, various methods have been used to measure ΔΨm based on either fluorescent probes or electrochemical methods. For example, rhodamine dyes (e.g., Rhodamine-123), carbocyanins, merocyanines, and oxonols have been used as fluorescent molecular probes to measure ΔΨm. Nano-electrodes used to impale the mitochondrial membrane in a patch clamp type assay are challenging. Many measurements to date have been based on assays of the distribution of lipophilic probe ions across the membrane, whose concentration ratio is related to ΔΨm through the Nernst equation. The probe ion concentration ratio is either measured through changes in fluorescence intensity (using cytofluorometry, confocal microscopy, fluoroescence microscopy, or fluoroescence spectroscopy) or electrochemically through ion selective electrodes (ISE).
Kamo et al. first reported an ISE membrane potential electrode using tetraphenylphosphonium (TPP+) ions, a lipid-soluble cation, and found that TPP+ can permeate through mitochondrial membranes with 15 times faster diffusion coefficient than other cations such as DDA+ (debenzyldimethyl ammonium). See N. Kamo, M. Muratsugu, R. Hongoh and Y. Kobatake, J. Membr. Biol., 1979, 49, 105-121. Since the accumulation of TPP+ ions into the mitochondrial matrix is related to ΔΨm through the Nernst equation and volumetric factors, its value can be determined from the concentration of TPP+ ions.
TPP+ ions diffuse through the mitochondrial inner membrane, the concentration ratio depending on ΔΨm, determined by the Nernst equation, i.e.
                                                        [              TPP              ]                        out                                              [              TPP              ]                        in                          =                  ⅇ                                    ΔΨ              m                        kT                                              (        1        )            
By measuring the concentration of TPP outside the mitochondria (referred to as “[TTP]out”) using electrochemical ion selective electrode technology one can infer the amount of cation taken up into the mitochondria, (hence termed “[TPP+]in”) to determine the membrane potential.
Several researchers reported improvements of the TPP+ selective electrode. Labajova et al. reported the construction of an optimized system for mitochondrial membrane potential measurement based on the TPP+-selective electrode discovered by Kamo. See A. Labajova, A. Vojtiskova, P. Krivakova, J. Kofranek, Z. Drahota and J. Houstek, Evaluation of mitochondrial membrane potential using a computerized device with a tetraphenylphosphonium-selective electrode, Anal. Biochem., 2006, 353, 37-42. Their device consisted of a measuring chamber with a maximum volume of 5 mL, reference electrode, TPP+-selective electrode, personal computer, and MATLAB/Simulink software that provided signal acquisition, processing, and display. Satake et al. reported a coated wire electrode that was sensitive to TPP+. Satake et al., Analytical Letters, 24(2), 295-304 (1991). Their device employed TPP+ tetraphenylborate as the ion sensor and was demonstrated to have a linear response for concentrations within 1 mM to 30 μM.
Current technology requires several hundred milligrams of isolated mitochondria for functional assays to determine ΔΨm. It is desirable, however, to have a functional assay that can operate with a much smaller sample of mitochondria. Because of very limited sample availability, technology advances that require reduced sample size (preferably, many orders of magnitude) will dramatically enable and/or facilitate the evaluation of mitochondrial function in clinical biopsy samples and certain cell lines.